Ana Amorim

Toxin composition of a Prorocentrum lima strain isolated from the Portuguese coast

Microalgae of the genus Dinophysis and Prorocentrum are known producers of okadaites, responsible for the human syndrome known as diarrhetic shellfish poisoning (DSP). In temperate regions, only species from the genus Dinophysis are commonly held responsible for shellfish contamination. This is probably related to the different ecological strategies of the two genera, namely the planktonic nature of Dinophysis versus the benthic/epiphytic nature of toxic Prorocentrum species. In recent years, the threat of global warming has drawn attention to the study of benthic toxic microalgae in southern European waters. Here we present results on the toxin production and toxin profile of a Prorocentrum lima strain isolated from the Portuguese coast. This strain, IO66-01, presented a mean growth rate of 0.49 divisions d(-1), not common in temperate strains, and only comparable with tropical strains. The parent toxins found were okadaic acid (OA) and dinophysistoxin-1 (DTX1). The major diol esters were D8- and D9- congeners of both OA and DTX1.

Protoperidinium minutum (Dinophyceae) from Portugal: cyst–theca relationship and phylogenetic position on the basis of single-cell SSU and LSU rDNA sequencing

Ribeiro S., Lundholm N., Amorim A. and Ellegaard M. 2010. Protoperidinium minutum (Dinophyceae) from Portugal: cyst–theca relationship and phylogenetic position on the basis of single-cell SSU and LSU rDNA sequencing. Phycologia 49: 48–63. DOI: 10.2216/09-11.1 Round brown spiny cysts are common elements of Recent and Quaternary dinoflagellate cyst records and are often used to infer past climate conditions. Echinidinium and Islandinium, two cyst-based genera composed of round brown spiny cysts, are believed to have affinities within the Protoperidiniaceae. However, their biological counterparts are still virtually unknown. In this study, we examined the cyst–theca relationship of an Echinidinium-like cyst isolated from recent sediments of the Portuguese coast. The cysts (25–34 µm) had an intercalary theropylic archeopyle and numerous processes (4–9 µm) with tapered stems and minutely expanded tips. Germinated cells were identified as Protoperidinium minutum on the basis of theca morphology and tabulation. This taxon has a complicated taxonomic history and most likely represents a complex of species with very similar thecae but different cyst morphologies. To provide a first step in elucidating the phylogeny of P. minutum and its evolutionary relationship among the Protoperidiniaceae, we undertook the first molecular study of this taxon on the basis of small-subunit (SSU) and large-subunit (LSU) ribosomal (r)DNA genetic sequences obtained through single-cell polymerase chain reaction. On the basis of SSU rDNA analysis, P. minutum formed a clade together with the Diplopsaloideae, not grouping together with the other Protoperidinium species. LSU rDNA-based phylogenies indicate P. minutum as early divergent within the Protoperidiniaceae. The evolutionary significance of round brown spiny cysts produced by P. minutum-like species and diplopsalids is discussed.

Historical cyst record as evidence for the recent introduction of the dinoflagellate Gymnodinium catenatum in the north-eastern Atlantic

The geographical origin of the warm-temperate paralytic shellfish poison producer Gymnodinium catenatum Graham is still under debate. It was first reported in the north-eastern Atlantic in 1976, from the Galician rías (North-West Iberia). Since then, and until 1995, recurrent blooms were recorded, with north-south progression of the affected areas, eventually affecting the whole West and South Atlantic coasts of Iberia and the Moroccan coast. This study presents results obtained from cyst analyses of a 210Pb-dated box core and a 14C-dated piston core, collected off Iberia, covering the last 2 000 years. They indicate that G. catenatum is not endemic to the area but has been introduced around the beginning of the last century, coinciding with another major environmental change, possibly related to cultural eutrophication.

Gymnodinium catenatum-like cysts (Dinophyceae) in recent sediments from the coast of Portugal

Abstract Gymnodinium catenatum has been responsible for the main paralytic shellfish poisoning (PSP) events reported along the Iberian coast, where much research effort has been put into understanding its bloom dynamics. Identification of a benthic resting stage in its life cycle raised questions regarding the implications of this life stage for bloom dynamics. When first described, the microreticulate cyst of G. catenatum was considered unique, but recently, two additional naked dinoflagellate species with different-sized microreticulate cysts have been described, viz. G. nolleri and G. microreticulatum. Here, we report on the size distribution of microreticulate cysts from recent sediments along the Portuguese coast and describe the occurrence of G. microreticulatum in European coastal waters. We also present field data on the distribution of G. catenatum cysts, which support the growing evidence for a planktonic origin for G. catenatum blooms in Iberian waters rather than in benthic cyst beds.

Reconstructing the history of an invasion: the toxic phytoplankton species Gymnodinium catenatum in the Northeast Atlantic

The phytoplankton species Gymnodinium catenatum is responsible for major worldwide losses in aquaculture due to shellfish toxicity. On the West coast of the Iberian Peninsula, toxic blooms have been reported since the mid-1970s. While the recent geographical spread of this species into Australasia has been attributed to human-mediated introduction, its origin in the Northeast Atlantic is still under debate. Gymnodinium catenatum forms a highly resistant resting stage (cyst) that can be preserved in coastal sediments, building-up an historical record of the species. Similar cyst types (termed microreticulate) are produced by other non-toxic Gymnodinium species that often co-occur with G. catenatum. We analysed the cyst record of microreticulate species in dated sediment cores from the West Iberian shelf covering the past ca. 150xa0years. Three distinct morphotypes were identified on the basis of cyst diameter and paracingulum reticulation. These were attributed to G. catenatum (35.6–53.3xa0μm), G. nolleri (23.1–36.4xa0μm), and G. microreticulatum (20.5–34.3xa0μm). Our results indicate that G. catenatum is new to the NE Atlantic, where it appeared by 1,889xa0±xa010, expanding northwards along the West Iberian coast. The earliest record is from the southernmost sample, while in the central Portuguese shelf the species appears in sediments dated to 1,933xa0±xa03, and in the North, off Oporto, in 1,951xa0±xa04. On the basis of the cyst record and toxic bloom reports, we reconstruct the invasive pathway of G. catenatum in the NE Atlantic. Although human-mediated introduction cannot be discarded, the available evidence points towards natural range expansion, possibly from NW Africa.

DISTRIBUTION AND GENETIC DIVERSITY OF THE LUCIFERASE GENE WITHIN MARINE DINOFLAGELLATES(1).

Dinoflagellates are the most abundant protists that produce bioluminescence. Currently, there is an incomplete knowledge of the identity of bioluminescent species arising from inter‐ and intraspecific variability in bioluminescence properties. In this study, PCR primers were designed to amplify the dinoflagellate luciferase gene (lcf) from genetically distant bioluminescent species. One of the primer pairs was “universal,” whereas others amplified longer gene sequences from subsets of taxa. The primers were used to study the distribution of lcf and assess bioluminescence potential in dinoflagellate strains representing a wide variety of taxa as well as multiple strains of selected species. Strains of normally bioluminescent species always contained lcf even when they were found not to produce light, thus demonstrating the utility of this methodology as a powerful tool for identifying bioluminescent species. Bioluminescence and lcf were confined to the Gonyaulacales, Noctilucales, and Peridiniales. Considerable variation was observed among genera, or even species within some genera, that contained this gene. Partial sequences of lcf were obtained for the genera Ceratocorys, Ceratium, Fragilidium, and Protoperidinium as well as from previously untested species or gene regions of Alexandrium and Gonyaulax. The sequences revealed high variation among gene copies that obscured the boundaries between species or even genera, some of which could be explained by the presence of two genetic variants within the same species of Alexandrium. Highly divergent sequences within Alexandrium and Ceratium show a more diverse composition of lcf than previously known.

Environmental change in the Western Iberia Upwelling Ecosystem since the preindustrial period revealed by dinoflagellate cyst records

We present the first multi-site study of dinoflagellate cyst records spanning ca. AD 1860–2000 from the west Iberian coast. Our aim was to reconstruct environmental changes in the Western Iberia Upwelling Ecosystem, one of the most biologically productive areas in the world, and an active fishery region. A major shift in cyst assemblages was recorded off the northwestern shelf sector between 1920 and 1950 towards autotrophic dominance, and consisted of a multi-fold increase in total cyst concentrations and cysts of Lingulodinium polyedrum, accompanied by an increase in Protoceratium reticulatum cysts. The observed changes pre-date the industrialization of agriculture in Portugal, and are concomitant with a shift in the North Atlantic Oscillation (NAO) towards negative indices (higher river input) and increasing sea-surface temperatures. The southernmost record, with reduced river- and human influence, showed only minor changes during the 20th century. Here, an increase in Protoperidinioid cysts after the 1980s is related to upwelling intensification. Our study indicates that the main changes recorded in the Western Iberian Upwelling system during the 20th century were driven by regional climate variability (warming, increased water stability and nutrient availability), possibly enhanced by anthropogenic nutrient input from the second half of the century. Our results highlight the complexity of the environmental drivers that may act upon dinoflagellate communities within the same region. The 20th-century environmental change in the Western Iberian Ecosystem has resulted in a shift towards marked autotrophic dominance of dinoflagellate cyst communities, and the northward expansion of species associated with Harmful Algal Blooms.

Gymnodinium catenatum Graham isolated from the Portuguese coast: Toxin content and genetic characterization

The bloom forming marine dinoflagellate Gymnodinium catenatum Graham has been linked to paralytic shellfish poisoning (PSP) outbreaks in humans. Along the Portuguese coast (NE Atlantic), G. catenatum shows a complex bloom pattern, raising questions about the origin and affinities of each bloom population. In this work, the variability within six cultured strains of G. catenatum isolated from Portuguese coastal waters (S coast, W coast and NW coast), between 1999 and 2011, was investigated. The strains were analyzed for toxin profiling and intra-specific genetic diversity. Regarding the toxin profile, differences recorded between strains could not be assigned to the time of isolation or geographical origin. The parameter that most influenced the toxin profile was the life-cycle stage that originated the culture: vegetative cell versus hypnozygote (resting cyst). At the genetic level, all strains showed similar sequences for the D1-D2 region of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA) and shared complete identity with strains from Spain, Algeria, China and Australia. Conversely, we did not find a total identity match for the ITS-5.8S nuclear rDNA fragment. After sequence analysis, two guanine/adenine (R) single nucleotide polymorphisms (SNP 1 and 2) were detected for all strains, in the ITS1 region. This species has been reported to present very conservative LSU and ITS-5.8S rDNA regions, though with few SNP, including SNP1 of this study, already attributed to strains from certain locations. The SNP here described characterize G. catenatum populations from Portuguese waters and may represent valuable genetic markers for studies on the phylogeography of this species.

The acritarchous resting cyst of the athecate dinoflagellate Warnowia cf. rosea (Dinophyceae)

Abstract Cysts of the athecate dinoflagellate Warnowia cf. rosea were found in surface sediment samples from the Norwegian coast near Bergen and from the coast of Portugal. The cyst has not been described before and is acritarch-like, in that the germination opening, a simple median split, is identical to chasmic openings found in fossil acritarchs. The cyst is 30–45 by 28–43 μm and light brown with irregular tufts of darker brown material and fused darker brown processes. The cyst often disintegrates rapidly after germination, and it is destroyed by palynological processing. Such a cyst would not be expected to persist in the fossil record. Nevertheless, its morphology supports the idea that athecate dinoflagellates are represented by acritarchs in the older geological record, predating the first morphologically identifiable dinoflagellate cysts.

Aquatic community structure in Mediterranean edge-of-field waterbodies as explained by environmental factors and the presence of pesticide mixtures

Studies addressing the predicted effects of pesticides in combination with abiotic and biotic factors on aquatic biota in ditches associated with typical Mediterranean agroecosystems are scarce. The current study aimed to evaluate the predicted effects of pesticides along with environmental factors and biota interactions on macroinvertebrate, zooplankton and phytoplankton community compositions in ditches adjacent to Portuguese maize and tomato crop areas. Data was analysed with the variance partitioning procedure based on redundancy analysis (RDA). The total variance in biological community composition was divided into the variance explained by the multi-substance potentially affected fraction [(msPAF) arthropods and primary producers], environmental factors (water chemistry parameters), biotic interactions, shared variance, and unexplained variance. The total explained variance reached 39.4% and the largest proportion of this explained variance was attributed to msPAF (23.7%). When each group (phytoplankton, zooplankton and macroinvertebrates) was analysed separately, biota interactions and environmental factors explained the largest proportion of variance. Results of this study indicate that besides the presence of pesticide mixtures, environmental factors and biotic interactions also considerably influence field freshwater communities. Subsequently, to increase our understanding of the risk of pesticide mixtures on ecosystem communities in edge-of-field water bodies, variations in environmental and biological factors should also be considered.